A recombinant type III humanized collagen, its preparation method and application

Abstract

This invention relates to the field of biotechnology, specifically to a recombinant type III humanized collagen, its preparation method, and its applications. The amino acid sequence of the recombinant type III humanized collagen is shown in SEQ ID NO.1. This invention provides a new sequence of recombinant type III humanized collagen, and has obtained a new recombinant type III humanized collagen. Cell experiments have demonstrated that the new recombinant type III humanized collagen obtained by this invention can significantly promote the proliferation and migration of human skin fibroblasts, meaning that the recombinant type III humanized collagen of this invention has stronger skin repair capabilities and possesses significant commercial application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a recombinant type III humanized collagen, its preparation method, and its application. Background Technology

[0002] Collagen is the most abundant structural protein in the human body. Type III collagen is mainly distributed in the skin, blood vessels, and internal organs, playing a crucial role in maintaining tissue elasticity and repairing and regenerating tissues. Currently, most collagen on the market is extracted from animal tissues (such as pig skin and bovine Achilles tendon), but this poses risks such as high immunogenicity (e.g., foot-and-mouth disease virus residue), pathogenic bacteria contamination, and rejection reactions caused by foreign proteins.

[0003] Although recombinant collagen technology avoids the risks associated with animal-derived products through genetic engineering, existing technologies still have significant drawbacks:

[0004] (1) Limitations in sequence design: Some recombinant collagens use non-humanized amino acid sequences (such as mouse collagen analogs), resulting in insufficient biological activity;

[0005] (2) Poor structural stability: Traditional Pichia pastoris expression systems are prone to producing misfolded proteins, leading to the destruction of the triple helix structure;

[0006] (3) Single function: Existing recombinant collagen lacks the ability to regulate cell behavior and is difficult to meet the needs of tissue regeneration.

[0007] Based on the shortcomings of existing technologies, this invention will develop a recombinant type III humanized collagen, its preparation method, and its applications. Summary of the Invention

[0008] The present invention first provides a recombinant type III humanized collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides biological materials related to the above-mentioned proteins, which are any one of B1) to B8) below:

[0010] B1) Nucleic acid molecules encoding the aforementioned recombinant type III humanized collagen;

[0011] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0012] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0013] B4) A recombinant vector containing the expression cassette described in B2);

[0014] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0015] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0016] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0017] B8) Recombinant microorganisms containing the recombinant vector described in B4);

[0018] Optionally, the vector includes a yeast expression vector;

[0019] Optionally, the recombinant microorganism is a yeast cell.

[0020] The present invention also provides the application of the above-mentioned recombinant type III humanized collagen in the preparation of drugs for skin repair, wound healing and scar reduction.

[0021] The present invention also provides the application of the above-mentioned biomaterials in the preparation of recombinant type III humanized collagen.

[0022] The present invention also provides a method for preparing recombinant type III humanized collagen, which includes the following steps: using the above-mentioned biological materials to obtain recombinant type III humanized collagen.

[0023] In some implementations, the sequence of the recombinant type III humanized collagen is shown in SEQ ID No. 1.

[0024] Finally, the present invention provides a product containing recombinant type III humanized collagen, the product comprising the above-mentioned protein or the above-mentioned biological material.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] This invention provides a novel recombinant type III humanized collagen sequence, and obtains a novel recombinant type III humanized collagen. Cell experiments have demonstrated that the novel recombinant type III humanized collagen obtained by this invention can significantly promote the proliferation and migration of human skin fibroblasts, which means that the recombinant type III humanized collagen of this invention has stronger skin repair capabilities and has great industrial application value. Attached Figure Description

[0027] Figure 1 A diagram showing how recombinant type III humanized collagen promotes cell proliferation.

[0028] Figure 2 A diagram illustrating how recombinant type III humanized collagen promotes cell migration. Detailed Implementation

[0029] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0030] A recombinant type III humanized collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0031] Biological materials related to the above proteins are any one of B1) to B8) below:

[0032] B1) Nucleic acid molecules encoding the aforementioned recombinant type III humanized collagen;

[0033] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0034] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0035] B4) A recombinant vector containing the expression cassette described in B2);

[0036] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0037] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0038] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0039] B8) Recombinant microorganisms containing the recombinant vector described in B4);

[0040] Optionally, the vector includes a yeast expression vector;

[0041] Optionally, the recombinant microorganism is a yeast cell.

[0042] The above-mentioned recombinant type III humanized collagen is used in the preparation of drugs for skin repair, wound healing and scar reduction.

[0043] The application of the above-mentioned biomaterials in the preparation of recombinant type III humanized collagen.

[0044] A method for preparing recombinant type III humanized collagen includes the following steps: using the above-mentioned biological materials, recombinant type III humanized collagen is obtained.

[0045] The sequence of the recombinant type III humanized collagen is shown in SEQ ID No. 1.

[0046] A product containing recombinant type III humanized collagen, the product comprising the aforementioned protein or the aforementioned biological material.

[0047] Example 1: Preparation of recombinant type III humanized collagen

[0048] (1) Gene synthesis and vector construction

[0049] This invention uses the amino acid sequence of the human type III collagen α1 chain published in the NCBI database as a template, and optimizes the nucleotide sequence of the amino acid sequence to obtain the amino acid sequence of SEQ ID No. 1.

[0050] SEQ ID No. 1:

[0051] gppgsngnpgppgpsgspgkdgppgpagntgapgspgvsgpkgdagqpgekgspgaqgppgapgplgiagitgarglagppgmpgprgspgpqgvkges gkpganglsgergargpqglpglagtagepgrdgnpgsdglpgrdgspggkgdrgengspgapgapghpgppgpvgpagksgdrgergargpagapgpa;

[0052] The nucleotide sequence encoding SEQ ID No. 1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and directionally cloned into the multiple cloning site (MCS) of the pUC57 plasmid. Specific primers with EcoRI / NotI double restriction sites were designed to amplify the target gene by PCR (reaction system: 50 μL, containing Phusion HF Master Mix, 0.5 μM primers each, and 100 ng template DNA). After separation by agarose gel electrophoresis (1% agarose, 1×TAE), the target band was recovered using a Gel Extraction Kit (Qiagen), which is the gene fragment encoding SEQ ID No. 1.

[0053] (2) Construction of Pichia pastoris expression system

[0054] The linearized pPIC9K expression vector (digested with SalI) was seamlessly ligated with the recovered gene fragment encoding SEQ ID No. 1 using the In-Fusion HD Cloning kit (Takara) to construct the recombinant plasmid pPIC9K-III-COL. After transformation into JM109 competent cells (TransGen Biotech), the plasmid was plated on LB plates containing 100 μg / mL Ampicillin and incubated at 37°C for 16 h. Single colonies were picked for colony PCR verification (primers: pPIC9K-F / R). Positive clones were verified by NotI digestion and sequencing (coverage >99%, GC content 42.3%), and the supercoiled plasmid (OD260 / 280 = 1.85) was extracted. 10 μg of the linearized plasmid was introduced into Pichia pastoris GS115 competent cells using electroporation (Gene Pulser Xcell™ system, 1.5 kV) and plated on MD solid medium (containing 100 μg / mL Zeocin). After gradient resistance screening (2-5 mg / mL G418), high-resistant colonies were selected for genomic PCR identification (primers: AOX1-F / R) to confirm that the recombinant plasmid was integrated into the AOX1 site of the genome.

[0055] (3) High-density fermentation and protein expression

[0056] The engineered strain was activated in YPD medium (30℃, 220 rpm, 16 h), and then transferred to BMGY medium (containing 1% yeast extract, 2% peptone, 1% glycerol, and 1.34% YNB) at a 1% inoculum. The culture was maintained at 30℃ until OD600≈20. During the induction phase, the medium was switched to BMMY medium (supplemented with 0.5% methanol), and 0.75% methanol was added every 24 h to maintain induced expression. Dissolved oxygen was controlled using an online DO-stat system (maintained >20% saturation), and the fermentation period was 96 h. SDS-PAGE analysis showed that the target band in the supernatant had a molecular weight of approximately 17.5 kDa (consistent with the theoretical value), and Coomassie Brilliant Blue quantification showed an expression level of 1.2 g / L.

[0057] (4) Two-step chromatography purification process

[0058] After sterilization via a 0.22 μm filter, the fermentation broth was initially purified using HiPrep™ DEAE FF anion exchange chromatography (GE Healthcare): the equilibration buffer was 20 mM Tris-HCl (pH 7.5), the elution gradient was 0–500 mM NaCl (flow rate 2 mL / min), and the 280 nm UV absorption peak was collected (purity >90%). Further fine purification was performed using a Source™ Q XL strong anion exchange column (Cytiva): the equilibration buffer was 20 mM Tris-HCl + 150 mM NaCl (pH 7.0), and linear gradient elution (0–1 M NaCl) yielded the target protein with a purity of 96.2% (SEC-HPLC determination, aggregate content <2%). Mass spectrometry analysis showed a molecular weight of 17.51 ​​kDa, consistent with the sequence characteristics of SEQ ID No. 1.

[0059] Example 2: Effects of recombinant type III humanized collagen on cell proliferation

[0060] Human skin fibroblasts (HSFs) in the exponential growth phase were selected at a ratio of 5 × 10⁻⁶. 4 Cells / wells were seeded at a density of 5 replicates per group in 96-well plates. After cell attachment, the original culture medium was removed, and a mixture of DMEM complete medium containing 10% fetal bovine serum and CCK-8 assay reagent (volume ratio 100:1) was added. The plates were incubated at 37°C in the dark for 2 hours. The reaction solution was then aspirated, and each well was rinsed once with pre-chilled PBS buffer. The experimental groups were cultured in DMEM medium containing recombinant human type III collagen (concentration gradient: 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL), while the control group received only fresh DMEM complete medium. After culturing for 96 hours, 100 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 2 hours. The absorbance (OD450) at 450 nm was measured using a microplate reader. Data analysis showed that the absorbance of the experimental groups increased in a dose-dependent manner with increasing collagen concentration. Figure 1 This indicates that the collagen can significantly promote the proliferation of HSF cells (P<0.01).

[0061] Example 3: Regulatory effect of recombinant type III humanized collagen on cell migration

[0062] After digesting and resuspending human skin fibroblasts HSF, they were subjected to 2×10⁻⁶... 4Cells / well were seeded at a density of [number] cells / well in the upper layer of Transwell® migration chambers, with the lower layer containing complete culture medium with 10% FBS as a chemokine. Different concentrations of recombinant type III collagen (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) were added to the culture medium in the experimental groups, while no exogenous collagen was added to the control group. The culture chambers were incubated at 37°C and 5% CO2 for 24 hours. After removing the culture chambers, unmigrated cells in the upper chamber were wiped off with a cotton swab, and the migrating cells on the bottom of the lower membrane were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.1% crystal violet for 30 minutes. Five high-power fields (×200) were randomly selected under a microscope, and the number of migrating cells was counted using ImagePro Plus software. The results showed that compared with the control group, the number of migrating cells in the 200 μg / mL collagen group increased by 4.8 times ([number]). Figure 2 This study confirmed that the recombinant humanized type III collagen enhances cell migration ability.

[0063] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant type III humanized collagen, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A biomaterial related to the recombinant type III humanized collagen of claim 1, comprising any one of B1) to B8) below: B1) The nucleic acid molecule encoding the recombinant type III humanized collagen of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4); The vector includes a yeast expression vector; The recombinant microorganism is a yeast cell.

3. The use of the recombinant type III humanized collagen according to claim 1 in the preparation of medicaments for skin repair, wound healing or scar reduction.

4. The application of the biomaterial according to claim 2 in the preparation of recombinant type III humanized collagen.

5. A method for preparing recombinant type III humanized collagen, characterized in that, It includes the following steps: Recombinant type III humanized collagen was obtained using the biomaterial described in claim 2.

6. The preparation method according to claim 5, characterized in that: The sequence of the recombinant type III humanized collagen is shown in SEQ ID No.

1.

7. A product containing recombinant type III humanized collagen, characterized in that, The product includes the protein as described in claim 1.

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